1. Field of the Invention
The present invention relates to a laser microscope and, more particularly, to a multi-photon excitation laser microscope using an ultra short pulse laser as excitation light.
2. Description of the Related Art
In the field of biology, a multi-photon excitation laser microscope is often used. This is so because a clear fluorescent image of a deep portion of a specimen can be obtained by tomography by using this multi-photon excitation laser microscope. On the other hand, when live cells are to be observed with a laser microscope, it is desirable to minimize the amount of laser beam with which the specimen is irradiated, in order to prevent damages to the specimen.
A general method of adjusting the amount of laser beam is to use an ND filter. A technique which stabilizes the illumination light amount and increases the repeatability of transmissivity is known (Jpn. Pat. Appln. KOKAI Publication No. 2000-19432).
To generate fluorescence by multi-photon excitation, multi-photons must be absorbed by a fluorescent dye of a specimen, but the multi-photon absorption probability is small. Therefore, the amount of laser beam must be increased to obtain a necessary fluorescence amount for observation. Unfortunately, if the amount of laser beam is increased, a fluorescent substance of a specimen readily causes photobleaching, and this makes long-time observation difficult. Also, this increases the possibility of the specimen being damaged by the laser beam energy.
Accordingly, in any multi-photon excitation laser microscope, the laser beam must therefore have a peak power large enough to achieve multi-photon absorption and an average power small enough to lessen the damage to specimens.
A relationship indicated byFn∝An∝In  (1)holds between an n-photon excitation fluorescence amount Fn, n-photon absorption probability An, and excitation light intensity I.
That is, when the excitation light intensity I is doubled, the n-photon excitation fluorescence amount Fn and probability An increase by 2n times. When the excitation light intensity I is halved, the n-photon excitation fluorescence amount Fn and probability An reduce by 1/2n times.
For example, if the laser beam intensity is reduced to 50% (=½) in order to suppress damages to a live cell, the excitation fluorescence amount becomes 25% (=¼) in 2-photon excitation. On the other hand, to perform observation with an excitation fluorescence amount of 50%, the laser beam intensity is set to about 70% in 2-photon excitation.